Project Summary: Blood vessels present a great barrier for delivering therapeutic nanoparticles, however the existing approaches are not sufficient to resolve this problem. Neutrophils are the most abundant white blood cells circulating in bloodstream, and are capable of transmigrating into tissues to rapidly respond tissue injury and infection. Therefore, neutrophils could be an excellent carrier to mediate nanoparticle transport across vessel barrier. Using intravital microscopy and an acute lung inflammation mouse model we have demonstrated that nanoparticles made from albumin protein can specifically bind activated neutrophils leading to the transport of these nanoparticles across endothelial barrier via neutrophil-mediated transmigration. Thus, we hypothesize that neutrophils can transport drugs loaded in albumin nanoparticles across a blood-vessel barrier and deposit the drugs in diseased tissues. Furthermore, using this novel approach we could prevent acute lung injury, a devastating disease currently without effective pharmacological therapies. We have proposed three aims: Aim 1: Define the properties of albumin nanoparticle platforms required for targeting of activated neutrophils in vivo. We will develop the methods to generate bright fluorescently-labeled albumin nanoparticles for tracking nanoparticles in cells and live mice. We will address the roles of particle size, density, surface charge and albumin properties in affecting nanoparticle uptake. We will determine drug loading efficiency in albumin nanoparticles and drug release profiles in vitro and in vivo using HPLC/mass spectroscopy. Aim 2: Dissect the mechanism of neutrophil-mediated nanoparticle transport across the blood- vasculature barrier. Using intravital microscopy of mouse cremaster tissues, we will real-time visualize the uptake of albumin nanoparticles by neutrophils and neutrophils carrying the nanoparticles transmigrate across endothelial vessels and move to inflammatory tissues. In a tracheal-LPS induced acute lung inflammation model, we will investigate whether nanoparticle uptake by neutrophils affects neutrophil biological functions (transport and activity) using confocal microscopy, biochemical approaches and knockout mouse models. Aim 3: Determine the efficacy of our nanoparticle platforms in treatment of acute lung injury. By measuring cytokines, neutrophil infiltration and plasma protein accumulation in mouse lungs, and histochemistry, we will evaluate whether albumin nanoparticles loaded with NF-?B inhibitors can dramatically mitigate lung inflammation/injury compared to the free drugs. At the end of these studies, we will develop the novel strategies of delivering therapeutic into targets based on neutrophil transmigration to diseased locations, and create a way to deliver nanoparticles across endothelial barrier. Our studies will change current strategies to treat inflammatory disorders underlying acute and chronic diseases including cancer. Specifically, our approach will significantly impact on the development of new pharmacological therapies to treat acute lung injury, and a severe form, acute respiratory distress syndrome (ARDS) because there are no effective therapies to treat these devastating diseases with 40% mortality. To enhance our research program, we request to update our intravital microscope via the funding opportunity of Administrative Supplement.